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e-Publications@Marquette

e-Publications@Marquette

Master's Theses (2009 -) Dissertations, Theses, and Professional Projects

Biomethane Production from Biodegradable Plastics

Biomethane Production from Biodegradable Plastics

Nicholas John Norio Benn

Marquette University

Follow this and additional works at: https://epublications.marquette.edu/theses_open

Part of the Engineering Commons

Recommended Citation Recommended Citation

Benn, Nicholas John Norio, "Biomethane Production from Biodegradable Plastics" (2019). Master's Theses (2009 -). 561.

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by

Nicholas J. N. Benn

A Thesis Submitted to the Faculty of the Graduate School, Marquette University,

In Partial Fulfillment of the Requirements For the Degree of Master of Science

Milwaukee, Wisconsin December 2019

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BIOMETHANE PRODUCTION FROM BIODEGRADABLE PLASTICS Nicholas J. N. Benn

Marquette University, 2019

Organic polymer plastics are often short-lived commodities for single-use that result in landfill buildup and persistence in the environment. Plastic waste accumulation can cause ecological damage. Plastic production continues to outpace plastic waste management and perpetuates the growing epidemic of plastic pollution. More efficient handling of plastics would be beneficial.

One improvement involves biodegradable plastics (i.e., bioplastics), particularly polylactic acid (PLA) and polyhydroxyalkanoates (PHA), which can alleviate

environmental concerns stemming from mismanagement. Yet, there are currently no bioplastic waste management strategies scalable to handle the millions of pounds of bioplastics that enter the waste stream. Therefore, new bioplastic resource recovery options were investigated through anaerobic co-digestion, a potential solution that can take advantage of existing digesters to convert bioplastic to biogas containing methane for renewable energy.

Bioplastics biodegrade, but their potential to completely biodegrade on a time-scale compatible with current anaerobic digestion technologies is largely unknown. Accordingly, base-catalyzed thermal pretreatments were investigated to increase

biodegradation rates. Batch experiments revealed pretreatments at 55 °C, pH 12 for PHAs and 90 °C regardless of pH for PLA produced the greatest increase in subsequent

bioconversion to methane. Polyhydroxybutyrate (PHB) showed the highest rate of methane recovery and was selected for high-rate anaerobic co-digestion investigations simulating full-scale anaerobic digestion at municipal water resource recovery facilities. Synthetic municipal primary solids were co-digested with untreated or pretreated PHB at a 15 d retention time and resulted in 79-93% and 84-98% bioplastic conversion to

methane, respectively, corresponding to a 5% additional increase when pretreated. Microbial communities analyzed via Illumina sequencing showed archaea were

unchanged in response to PHB co-digestion, whereas the bacterial community changed, with increased relative abundance of Kosmotoga, Deferribacter, Geobacter, and

Ruminococcus. Therefore, these taxa may be important for PHB biodegradation.

The results of the current study suggest anaerobic co-digestion at municipal water resource recovery facilities is a feasible waste management option for PHB bioplastics, which may help to alleviate challenges associated with contemporary single-use plastics. Near complete conversion of PHB bioplastic to methane in just over two weeks signals a great compatibility with completely-stirred tank reactor co-digestion.

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ACKNOWLEDGEMENT

Nicholas J. N. Benn

First and foremost, I would like to sincerely thank Dr. Daniel H. Zitomer for graciously serving as my adviser and mentor during my undergraduate and graduate career at Marquette University. I’m boundlessly grateful for his patience, accessibility, encouragement, and insightfulness throughout all of my academic endeavors. His wisdom in both academia and life have been invaluable to me and my own personal development. His contributions of original ideas, time, connections and funding were vital to making this project a success. It was a great honor to work under his supervision, and I cannot stress enough how grateful I am for his mentorship and generosity. To my committee – Dr. Brooke Mayer, Dr. Patrick McNamara, and Dr. Kaushik Venkiteshwaran – I’m grateful for your time and willingness to participate in this project.

A special thanks is owed to Dr. Anne Schauer-Gimenez, Dr. Margaret Morse, and Mango Materials Inc. for their tireless work in the realm of biodegradable plastics, providing test materials, and efforts to facilitate a fruitful research collaboration,

including a summer internship at their United States Department of Agriculture (USDA) hosted laboratory in Albany, CA. I am especially grateful for the tremendous effort, insight, and friendship of Dr. Anne Schauer-Gimenez throughout this collaboration, her consummate energy and positivity during conference calls were unmatched. I’m indebted to the Water Equipment and Policy Center (WEP), a National Science Foundation (NSF) Industry – University Cooperative Research Centers (IUCRC) site, for providing two

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years of research funding. I also thank the Virginia Institute of Marine Science for providing bioplastic samples.

I’m appreciative for the ongoing assistance, both in the lab and office, from Dr. Kaushik Venkiteshwaran and value his friendship. His expertise in microbial community analysis were vitally important in deciphering the anaerobic digester microbiome. Thanks also to Julián Velazquez, Dylan Friss, Seyedehfatemeh (Saba) Seyedi, and Dr. Daniel Carey for lending a helping hand with lab work. Their assistance allowed me a break from research and gave me much needed time for travel and outdoor recreations. Mike Dollhopf’s aptitude for managing a laboratory is not without notice and his efforts keep the day-to-day operations running smoothly. Tom Silman’s assistance in the Marquette University opus College of Engineering Discovery Learning Center is much appreciated and his work to construct our lab digesters was vital to this and future projects’ successes. Dave Newman’s allowance to use his equipment was very helpful for processing

bioplastics and my arms are especially grateful. To everyone in the Water Quality Center, I am so happy to have crossed paths and have enjoyed our shared experiences together. The Water Quality Center and Graduate School’s financial support for travel awards to the Water Environment Federation Technical Exposition and Conference (WEFTEC) in

New Orleans and the International Water Association 16th World Congress on Anaerobic

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DEDICATION

I would like to dedicate this thesis to my parents, Perry Benn and Cheryl Iwami-Benn, who have been a constant source of motivation and provision. Their guidance has taught me the value of hard work and persistence and has been vital in my life and academic pursuits. I would also like to thank the Gerndt brothers and Sabrina for their encouragement and emotional support throughout this project.

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TABLE OF CONTENTS

ACKNOWLEDGEMENT ... i

DEDICATION ... iii

LIST OF TABLES ... viii

LIST OF FIGURES ... ix

1 INTRODUCTION ... 1

1.1 Motivation ... 1

1.2 Hypotheses & Objectives ... 2

1.3 References ... 3

2 LITERATURE REVIEW ... 4

2.1 Anaerobic digestion and co-digestion of PHA ... 4

2.2 Microbial community composition of anaerobic PHA degrading microbes ... 6

2.3 References ... 9

3 PRETREATMENT and ANAEROBIC CO-DIGESTION of SELECTED PHB and PLA BIOPLASTICS ... 13

3.1 Abstract ... 14

3.2 Introduction ... 15

3.3 Materials and Methods ... 18

3.3.1 Bioplastics ... 18

3.3.2 Bioplastics Processing and Pretreatment ... 21

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3.3.4 Anaerobic Co-digesters ... 23

3.3.5 Analyses... 25

3.4 Results and Discussion ... 26

3.4.1 Bioplastic Pretreatment and BMP Assays ... 26

3.4.2 Bench Scale Co-digestion ... 33

3.5 Conclusions ... 37

3.6 Acknowledgments ... 37

3.7 References ... 37

4 METHANE YIELD and LAG CORRELATE with BACTERIAL COMMUNITY SHIFT FOLLOWING PHB BIOPLASTIC ANAEROBIC CO-DIGESTION ... 41

4.1 Abstract ... 42

4.2 Introduction ... 42

4.3 Material and Methods... 46

4.3.1 Bioplastic Processing and Pretreatment ... 46

4.3.2 Anaerobic Co-Digesters ... 47

4.3.3 DNA Extraction and Illumina Sequencing Analyses ... 49

4.3.4 Major, Minor and Significant OTUs ... 49

4.3.5 Microbial Community Analyses ... 50

4.3.6 Anaerobic digester Performance Analyses ... 51

4.4 Results and Discussion ... 52

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4.4.2 Microbial Community Analyses ... 55

4.4.2.1 Major Bacterial OTUs ... 57

4.4.2.2 Major Archaeal OTUs ... 58

4.4.2.3 Spearman Correlation to Select Significant OTUs ... 59

4.4.3 The Role of Positively Correlated OTUs in Anaerobic PHB Degradation ... 66

4.5 Conclusions ... 69

4.6 Acknowledgements ... 69

4.7 References ... 69

5 Overall Conclusions and Recommendations ... 76

Appendices ... 80

Appendix 3 ... 80

Figure 3A Bench scale co-digestion pH ... 80

Figure 3B Bench scale co-digestion VFAs... 81

Table 3A Basal nutrient media ... 82

Table 3B PHB1 BMP results ... 83

Table 3C PHB2 BMP results ... 85

Table 3D PHB3 BMP results ... 86

Table 3E PHB4 BMP results ... 87

Table 3F PLA BMP results ... 88

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Appendix 4 ... 90

Table 4A Co-digestion functional meta data ... 91

Table 4B ANOSIM data values ... 92

Figure 4A Alpha diversity indices versus methane production rate ... 93

Figure 4B Major (A) bacterial OTUs and (B) archaeal OTUs community comparison ... 94

Figure 4C Relative abundance heatmap with dual hierarchical clustering of 30 significant OTUs... 95

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LIST OF TABLES

Table 3.1 Summary of Bioplastics ... 20 Table 3.2 Bench scale digestion and co-digestion meta data ... 34 Table 43.1 Spearman correlation with methane production indicating 30 significant OTUs ... 60

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LIST OF FIGURES

Figure 1.1 Circular lifecycle for PHB bioplastics and methane ... 2 Figure 3.1 Scanning electron micrographs of untreated and pretreated PHB2 (MirelTM

F1006) ... 27

Figure 3.2 BMP values for untreated and pretreated bioplastics under conditions

resulting in the greatest biomethane increase ... 28

Figure 3.3 Average cumulative biomethane produced during BMP assays ... 32 Figure 3.4 Daily biomethane production for continuously fed anaerobic digesters ... 36 Figure 4.1 Digester average methane production co-digesting with synthetic municipal

primary sludge (SMPS) and PHB ... 53

Figure 4.2 Digester microbial communities comparison NMDS plots during pre- and

post-co-digestion periods ... 56

Figure 4.3 Digester microbial communities comparison NMDS plots during pre-,

transition-, and post-co-digestion periods ... 63

Figure 4.4 Analysis of similarity for pre- vs transition- and transition- vs

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1 INTRODUCTION 1.1 Motivation

Most plastic waste is non-biodegradable and causes environmental problems. A potential solution relies on new, biodegradable plastics. A cradle-to-cradle scenario involves anaerobic digesters in which bioplastic may be converted to biomethane (Figure 1.1). Bioplastics tested include polyhydroxybutyrate (PHB) and polylactic acid (PLA). We propose to develop a new pretreatment and anaerobic digestion process to convert bioplastics to biomethane for renewable energy. Processing and pretreatments required for rapid anaerobic digestion of bioplastics, their biomethane yields, and microbial community compositions have not been previously determined to the author’s knowledge.

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Figure 1.1 Circular lifecycle for PHB bioplastics and methane with a focus on step 5,

biologically converting post-consumer bioplastics back to methane through anaerobic digestion (inspired by Rostkowski et al., 2012).

1.2 Hypotheses & Objectives

The following three hypotheses with associated research objectives were investigated:

(1) Base-catalyzed thermal pretreatment is necessary to render bioplastics amenable to digestion in the time scale of anaerobic digestion. Research objectives associated with this hypothesis were as follows:

 Develop bioplastic preprocessing protocol to establish uniform particle size  Develop bioplastic liquid suspension base-catalyzed thermal pretreatment

protocol for conditions at pH 7, 8, 10, and 12, temperatures at 35, 55, and 90 °C, and incubation time for 3, 24, and 48 hours.

 Screen each bioplastic temperature and incubation time pretreatment profiles with standardized biochemical methane potential (BMP) tests to identify optimum pretreatment profiles for increased biomethane yield.

 Screen pH conditions at the two most optimum pretreatment temperature profiles at all three incubation times with BMP tests to identify the optimum pretreatment conditions for increased biomethane yield. The most promising pretreatment profile of two PHB bioplastics are then used for bench-scale co-digestion investigations.

(2) Continuously fed, bench-scale co-digestion of pretreated PHB bioplastics will increase the biomethane yield compared to that of untreated PHB. Research objectives associated with this hypothesis were as follows:

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 Prior to PHB co-digestion, quasi-steady state continuously fed anaerobic digesters treating a synthetic municipal primary sludge (SMPS) will establish consistent digester performance and microbial communities. This provides a baseline for comparison to PHB co-digestion.

 Following SMPS digestion, untreated and pretreated PHB was continuously co-digested until quasi-steady state to evaluate daily biomethane yield due to PHB and impact of pretreatment on the rate and extent of biomethane production. (3) Feeding PHB as an anaerobic co-substrate will select microbial communities enriched

for hydrolytic and fermentative bacteria, catalyzing the initial breakdown of polymeric substances, but have little impact on archaea. Research objectives associated with this hypothesis were as follows:

 Illumina sequencing of the highly conserved region of the 16S rRNA gene from pre-, transition, and post- PHB co-digestion phases will show relative abundance changes as co-digesters acclimate from SMPS substrate alone to addition of PHB.

1.3 References

Rostkowski, K.H., Criddle, C.S., Lepech, M.D., 2012. Cradle-to-gate life cycle

assessment for a cradle-to-cradle cycle: Biogas-to-bioplastic (and back). Environ. Sci. Technol. https://doi.org/10.1021/es204541w

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2 LITERATURE REVIEW

2.1 Anaerobic digestion and co-digestion of PHA

Anaerobic biodegradation studies of PHAs began in the 1980s when bioplastics began to be developed on an industrial scale for single-use commodity applications, like plastic beverage bottles (Holmes, 1985; Stieb and Schink, 1984). Previous, early studies laid the groundwork for future biodegradation studies by establishing fundamental knowledge and showing that PHAs are a naturally occurring microbial carbon storage polyester that is readily biodegradable. PHB and a related copolymer,

poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), were studied with batch tests, pure culture plates, or enzymatic assays to determine their biodegradability over a defined period or until complete mineralization had taken place. Anaerobic degradability studies of PHAs primarily investigated inocula from anaerobic digesters at industrial or

wastewater treatment plants (Budwill et al., 1992; Gartiser et al., 1998; Mergaert and Swings, 1996; Reischwitz et al., 1998; Yagi et al., 2014, 2013, 2009), various

environmental sources, like pond sediments, rumen fluid, and spring water, (Budwill et al., 1996) as well as pure cultures (Janssen and Schink, 1993). Numerous PHA

biodegradability studies utilized aerobic inocula from soils and other environmental sources (Brandi et al., 1995; Jendrossek et al., 1996; Mergaert et al., 1994, 1993; Schink et al., 1992), while one study named approximately 700 different microbial strains encompassing 59 different taxa that could degrade PHB (Mergaert and Swings, 1996).

Anaerobic biodegradation studies of bioplastics would resume, spurred by the emergence of a newly-available bioplastic called polylactic acid (PLA), for which usage has increased worldwide due to cost reductions from cheap feedstocks, technology

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maturity, and economy of scale (Gross and Kalra, 2002; Muller et al., 2017). In 2018, it was estimated that 2.33 million tons of bioplastics were produced, 10.3% comprised PLA, nearly 240,000 tons, whereas PHAs accounted for 1.4%, approximately 32,600 tons (European Bioplastics, 2018). PLA bioplastic is different than PHA’s in that the

monomer, lactic acid, is produced through microbial fermentation and then polymerized through a series of industrial chemical processes (Lunt, 1998). PLA in its polymer form is not a microbial product and some anaerobic degradability tests have shown that it does not degrade as quickly nor yield as much biomethane compared to PHAs (Narancic et al., 2018). Yagi et al. (2009, 2013, 2014) found that PLA only began to degrade after 55 days at mesophilic temperatures to achieve up to 22-49% degradation within 277 days and required thermophilic conditions to reach degradation of 82-90% within 96 days. Criddle et al. (2014) similarly found that biogas generation from PLA was delayed approximately 35 days and biogas was nearly double after 120 days of incubation during thermophilic conditions compared to mesophilic conditions. Kolstad et al. (2012) and Vargas et al. (2009) also showed high rates of PLA degradation and biomethane yield during thermophilic digestion, 40-80% within 60 days. All other reports of anaerobic

biodegradation of PLA at mesophilic temperatures revealed poor biomethane production or weight loss within 60-390 days of tests (Gartiser et al., 1998; Vargas et al., 2009; Endres and Siebert-Raths, 2011; Kolstad et al., 2012; Krause and Townsend, 2016; Narancic et al., 2018). However, PLA will degrade during industrial composting in which aerobic conditions cause high temperatures stemming from rapid biodegradation of organic matter.

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Numerous studies have investigated PHAs as a component of municipal or industrial anaerobic digestion (Morse et al., 2011; Huda et al., 2013; Yagi et al., 2013, 2014; Soda et al., 2016; Wang et al., 2015, 2016, 2018; Narancic et al., 2018; Sethupathy and Sivashanmugam, 2018). A majority of these studies focused on batch anaerobic digestion tests that do not simulate operations that occur during typical continuous-fed digestion, and found that approximately 60 – 100% of PHAs were converted to

biomethane. However, only one study briefly looked into continuously-fed co-digestion to analyze archaeal relative abundance, but this special case of intracellular PHAs within waste activated sludge organisms was studied and not the usable form of bioplastic (Wang et al., 2015). Wang et al. (2015) co-digested waste activated sludge containing PHA in the range of 21 (± 4) to 184 (± 16) mg PHA/g VSS (volatile suspended solids). The results of these studies indicate that even small amounts of PHA can rapidly increase biomethane production from anaerobic digestion.

The work described in this thesis focused on anaerobic digestion of exogenous PHA from commercial sources because its application is intended to degrade post-consumer PHAs and maximize biomethane production. Previous investigations have indicated that PHA can be anaerobically biodegraded and co-digested, whether the PHA was intracellular and at low OLR or exogenous PHA at much higher OLR.

2.2 Microbial community composition of anaerobic PHA degrading microbes

The understanding of biodiversity of PHA degrading microbes is developed for aerobic microbes, but anaerobic-correlated PHA degrading microbes have not been as

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thoroughly studied (Mergaert and Swings, 1996). Studies conducted in the 1980s found that newly discovered anaerobic microbes could degrade hydroxybutyrate, the monomer comprising PHB (i.e., Ilyobacter polytropus (Stieb and Schink, 1984)) and a unique syntrophic bacterium, Syntrophomonas wolfei (McInerney et al., 1979; Wofford et al.,

1986), that can grow when a H2-utilizing microbe like a hydrogenotrophic methanogen is

present. Two anaerobic microbes that can degrade PHB were found by pure culturing methods in the 1990s, Ilyobacter delafieldii (Janssen and Harfoot, 1990; Janssen and Schink, 1993) and a bacterium from Clostridium group I (strain LMG 16094) (Mergaert et al., 1996). Most of these early studies relied upon culturing techniques, gram staining, and microscopic analysis to characterize microbes.

Within the last few years, modern DNA sequencing technologies have allowed researchers to characterize more anaerobic microbes responsible for anaerobic PHA degradation. The report by Wang et al. (2018) was the only study found that utilized 16S rRNA gene Illumina sequencing technology for microbial community analysis of

methanogenic PHA degrading batch tests. However, sample preparation was

unconventional for anaerobic digesters, centrifuged digestate supernatant was filtered and membranes frozen, which may not have accurately reflected the microbial community. Bacterial orders Cloacamonales, Thermotogales, and two unidentified taxa were enriched, whereas archaea were not discussed.

Yagi et al. (2014) performed batch anaerobic digestion tests of PHB under mesophilic conditions with inoculum from an industrial anaerobic digester fed cow manure and vegetable waste and found eubacteria of an uncultured strain of Clostridium and Arcobacter thereius with low-level detection of archaeal strains including

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Methanobacterium petrolearium, Methanobacterium sp (uncultured strain), and Methanosaeta concilii (Yagi et al., 2014). Yagi et al. (2013) similarly performed batch

anaerobic digestion of PHB at thermophilic conditions and found eubacteria strains

Peptococcacea bacterium Ri50, Bacteriodes plebeius, and Catenibacterium mitsuokai

with no archaeal strains described. Yagi et al. (2013) also found different bacteria

responsible for anaerobic digestion of three biopolymers together (PHB, PLA, and PCL – polycaprolactone), including Bacillus infernus, Propioni bacterium sp, and two

uncultured strains; no mention of archaeal strains was made. The Yagi et al. (2013, 2014) studies utilized RNA extraction, reverse transcription-polymerase chain reaction

amplification (RT-PCR), denaturing gradient gel electrophoresis (DGGE) profiles, and Sanger sequencing to detect and identify taxa based on their 16s rRNA sequence. Wang et al. (2015) operated semi-continuously fed anaerobic co-digesters to biodegrade WAS with intracellular PHA for 90 days. They investigated the relative abundance of archaea with a WAS feed containing low levels of PHA (21 mg PHA/g VSS) and high levels of PHA (184 mg PHA/g VSS) and found 34.5 ± 4.2% and 52.6 ± 5.7% archaeal abundance, respectively, based on 16s rRNA gene fluorescence in situ hybridization (FISH).

Conversely, the Yagi et al. (2013) study described low detection of archaea, albeit their methods were not quantitative, whereas Wang et al. (2015) found very high abundance values of archaea, which may indicate inconclusive results and method bias, in terms of archaeal communities. The microbial communities and key microbial taxa involved in anaerobic digestion and co-digestion of PHAs, especially archaea, requires further investigation.

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2.3 References

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Budwill, K., Fedorak, P.M., Page, W.J., 1996. Anaerobic microbial degradation of poly (3-hydroxyalkanoates) with various terminal electron acceptors. J. Environ. Polym. Degrad. 4, 91–102. https://doi.org/10.1007/BF02074870

Budwill, K., Fedorak, P.M., Page, W.J., 1992. Methanogenic degradation of poly(3-hydroxyalkanoates). Appl. Environ. Microbiol. 58, 1398–401.

Criddle, C.S., Billington, S.L., Frank, C.W., 2014. Renewable Bioplastics and

Biocomposites From Biogas Methane and Waste-Derived Feedstock: Development of Enabling Technology, Life Cycle Assessment, and Analysis of Costs, California Department of Resources Recycling and Recovery.

Endres, H.-J., Siebert-Raths, A., 2011. End-of-Life Options for Biopolymers, in: Engineering Biopolymers. Carl Hanser Verlag GmbH & Co. KG, pp. 225–243. https://doi.org/doi:10.3139/9783446430020.006

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Holmes, P.A., 1985. Applications of PHB-a microbially produced biodegradable thermoplastic. Phys. Technol. 16, 32–36.

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2018. Biodegradation of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Plastic under Anaerobic Sludge and Aerobic Seawater Conditions: Gas Evolution and Microbial Diversity. Environ. Sci. Technol. 52, 5700–5709.

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biodegradation test and analysis of eubacteria and archaea involved in anaerobic biodegradation of four specified biodegradable polyesters. Polym. Degrad. Stab. 110, 278–283.

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3 PRETREATMENT and ANAEROBIC CO-DIGESTION of SELECTED PHB and PLA BIOPLASTICS

This chapter has been published in the journal Frontiers in Environmental Science as:

Benn, N., Zitomer, D., 2018. Pretreatment and anaerobic co-digestion of selected PHB and PLA bioplastics. Front. Environ. Sci. 5. https://doi.org/10.3389/fenvs.2017.00093.

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3.1 Abstract

Conventional petroleum-derived plastics are recalcitrant to biodegradation and can be problematic as they accumulate in the environment. In contrast, it may be possible to add novel, biodegradable bioplastics to anaerobic digesters at municipal water resource recovery facilities along with primary sludge to produce more biomethane. In this study, thermal and chemical bioplastic pretreatments were first investigated to increase the rate and extent of anaerobic digestion. Subsequently, replicate, bench-scale anaerobic co-digesters fed synthetic primary sludge with and without PHB bioplastic were maintained for over 170 days. Two polyhydroxybutyrate (PHB), one poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and one polylactic acid (PLA) bioplastic were investigated.

Biochemical methane potential (BMP) assays were performed using both untreated bioplastic as well as bioplastic pretreated at elevated temperature (35–90 °C) under alkaline conditions (8<pH<12) for 3–48 h. PHB and PLA pretreatment increased average BMP values to over 100%. Average PHB lag time before methane production started, decreased when pretreatment was performed. Bench-scale anaerobic co-digesters fed synthetic primary sludge with PHB bioplastic resulted in 80–98% conversion of two PHB bioplastics to biomethane and a 5% biomethane production increase compared to

digesters receiving untreated PHB at the organic loadings employed (sludge OLR = 3.6 g

COD per L of reactor volume per day [g COD/LR-d]; bioplastic OLR = 0.75 g theoretical

oxygen demand per L of reactor volume per day [ThOD/LR-d]). Anaerobic digestion or

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3.2 Introduction

Conventional plastics derived from petroleum are not biodegradable to a significant extent and result in accumulation of plastic waste in landfills or natural environments (Rostkowski et al., 2012). Conventional plastics accumulate most notably in oceans where they have been shown to disintegrate, forming microplastic particles that adsorb pollutants such as polychlorinated biphenyls (PCBs), pesticides, and phthalates (Andrady, 2011). Microplastic particles with sorbed pollutants can be consumed by marine organisms and enter the human food chain (Hammer et al., 2012; Mato et al., 2001).

To be considered biodegradable, bioplastics must exceed 90% carbon conversion to carbon dioxide during aerobic composting within 180 days (Brodhagen et al., 2017; Narancic et al., 2018). Polyhydroxybutyrate (PHB) bioplastic is biodegraded in aerobic and anaerobic engineered processes as well as natural environments; however anaerobic co-digestion of PHB for the express purpose of waste management and renewable energy has not been investigated (Abou-Zeid et al., 2004; Deroiné et al., 2014; Gómez and Michel, 2013; Volova et al., 2010). Budwill et al. (1996) reported that PHB is

anaerobically biodegradable in various scenarios and suggested that municipal anaerobic sewage sludge digesters were suitable PHB degrading environment to generate

biomethane. PHB was shown to anaerobically biodegrade over 90% in 10 days at mesophilic conditions, whereas polylactic acid (PLA) only biodegraded 7% in 90 days even though it is considered to be industrially compostable under aerobic thermophilic conditions (Yagi et al., 2014). Despite lesser biodegradability, PLA is more readily

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available on the market today due to more efficient production at full scale (Gómez and Michel, 2013; Kolstad et al., 2012; Yagi et al., 2014, 2013).

To help mitigate the environmental concerns of conventional plastics, a more efficient coupling of bioplastic production and waste management should be developed (Gironi and Piemonte, 2011). According to cradle-to-gate lifecycle assessments (LCA), the biodegradable bioplastic PHB has potentially lower ecological impacts and global warming potential than conventional plastics if feedstocks are biobased and originate as by-products or wastes (Narodoslawsky et al., 2015). Other LCA researchers investigated PHB in a more holistic cradle-to-cradle scenario profiling an optimized process scheme with the assumption of complete biomethane recovery using anaerobic biodegradation and concluded that PHB was superior to conventional plastic in terms of global warming potential (Rostkowski et al., 2012). The assumption for complete biomethane recovery was described as an end of life option in which PHB was converted to biogas at an

anaerobic digestion facility. Direct evidence supporting anaerobic digestion of bioplastics such as PHB to biomethane in a waste management scenario is limited. Anaerobic

digestion feasibility is often assumed with results from anaerobic batch tests that may not accurately reflect operation of continuously fed digesters at quasi steady state.

Waste management and renewable energy generation from some biodegradable bioplastics could be achieved through anaerobic co-digestion using existing infrastructure and minimal process modification. With co-digestion, two or more feed materials, such as biodegradable plastic and municipal primary sludge, are fed to an anaerobic digester concomitantly. Co-digestion is implemented at some existing municipal water resource recovery facilities that often have excess capacity as well as boilers and

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electricity-generating equipment that employ biomethane (Navaneethan et al., 2011). Onsite storage of bioplastics, like PHB, could supplement anaerobic digestion by providing a dense source of carbon that may be utilized to blend with other influent waste streams. PHB has a bulk theoretical oxygen demand (ThOD) of 2,200 g ThOD/L, whereas synthetic

municipal primary sludge contains approximately 50 g COD/L. In addition, Stroot et al. (2001) suggested a C:N ratio for anaerobic digestion in the range of 20:1–30:1, but municipal sewage sludge for digestion was found to have C:N ratios ranging from 6:1 to 16:1, whereas the bioplastics contain C, but no N. Thus, co-digestion of bioplastics can increase C:N ratio to suggested values as well as result in increased biomethane

production for renewable energy generation.

Bioplastics, like PHB and PLA encountered in the consumer market, are water insoluble, hydrophobic polyesters that can be hydrolyzed by water-soluble endogenous carboxylesterase enzymes secreted by microbes. Carboxylesterases, like PHA

depolymerase or lipase, disrupt the ester linkages between bioplastic monomers and release them from bioplastic as water soluble molecules becoming bioavailable for microbial metabolism (Yoshie et al., 2002). An obligate anaerobic bacterium, Ilyobacter

polytropus, was evaluated in pure culture and was found to ferment 3-hydroxybutyrate to

acetate and butyrate (Stieb and Schink, 1984). In order to facilitate more rapid bioplastic transformation to biomethane on the time scale of municipal anaerobic digestion, the surface area could be increased through chemical and thermal processing and

pretreatment. Abiotic hydrolysis or depolymerization of PHA bioplastics into monomeric constituents and intermediate breakdown products was demonstrated at a pH of 13 in 0.1

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M sodium hydroxide aqueous solution at temperatures ranging from 60 to 70 °C and various incubation periods (Yu et al., 2005).

Over 70% abiotic degradation of PHB was demonstrated at 70 °C in 4 M sodium hydroxide after 4 h of treatment. Treatment of PHB in acidic solutions of sulfuric acid (0.05–2 M) at 70 °C for up to 14 h did not result in abiotic degradation (Yu et al., 2005). Near complete abiotic degradation of the copolymer PHBV was shown at 60 °C in 0.1 M sodium hydroxide after 18 h of treatment (Myung et al., 2014b). Thus, pretreatment in alkaline media at elevated temperatures induced polyester backbone hydrolysis resulting in release of water soluble breakdown products such as 3-hydroxybutyrate and crotonate, which have both been shown to support growth of strictly anaerobic microbes (Dörner and Schink, 1990; Janssen and Harfoot, 1990).

In this study, bioplastic thermal and chemical pretreatments were employed to increase the rate and extent of anaerobic digestion and co-digestion of commercially available PHB and PLA bioplastics. In order to elucidate the applicability of bioplastic pretreatments for anaerobic digestion and co-digestion, biochemical methane potential (BMP) assays were performed and methane yields were compared. Bench-scale anaerobic co-digestion of two PHB bioplastics, both pretreated and untreated, at quasi steady state with synthetic municipal primary sludge was then performed.

3.3 Materials and Methods 3.3.1 Bioplastics

Bioplastics tested include four PHB varieties including one

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Y3000 powder and MirelTM F1006 bioplastics were produced through fermentation of

D-glucose. The PHB copolymer MirelTM M2100 (4.4% 4-hydroxybutyrate) was produced

through fermentation of D-glucose and 1, 4-butanediol. PHB produced by Mango

Materials, Inc. was made from biomethane from an anaerobic digester. The PLA IngeoTM

2003D was obtained from a commercial, cold drink cup and may have contained other proprietary additives not reported by the manufacturer; this bioplastic was produced by fermentation of corn-derived dextrose followed by polymerization.

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20

(Manufacturer) (°C)

ENMAT™ Y3000 PHB1 PHB 176, NA Powder

(TianAn Biologic Materials Co.)

Mirel™ F1006 PHB2 PHB 165, 123 Pellet

(Metabolix, Inc. & Telles LLC a) (thermo formed)

Methane-derived bioplastic PHB3 PHB 172, NA Powder

(Mango Materials, Inc.)

Mirel™ M2100 PHB4 PHB 169, NA Pellet

(Metabolix, Inc. & Telles LLC a) [4.4% 4-HB] (extruded)

Ingeo™ 2003D PLA PLA 145, 55 Cup

(NatureWorks LLC) (thermo formed)

a Manufacturing discontinued b Melting temperature

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3.3.2 Bioplastics Processing and Pretreatment

Bioplastics were processed using methods similar to those reported by others (Witt et al., 2001; Yagi et al., 2013). Briefly, pelletized or thermoformed bioplastic samples were immersed in a liquid nitrogen bath for approximately 5 min to make them brittle and easier to grind, mechanically ground in a laboratory blender with a stainless steel canister (Waring 700G Commercial Blender), and sieved to less than 0.15 mm particle size. All bioplastics evaluated, apart from methane-derived PHB manufactured by Mango Materials, were commercially available at the time of testing. The Mango Materials plastic was obtained from the manufacturer as a prototype sample that was not yet commercially available. The commercially available bioplastics contain additives such as plasticizers and inks that may have influenced anaerobic digestion results.

Processed bioplastics were pretreated to increase surface area or initiate

depolymerization to facilitate increased biomethane evolution during anaerobic digestion and co-digestion. Pretreatments were performed for each bioplastic using two methods. The first method involved only thermal pretreatment. This was done at 35, 55, and 90 °C for 3, 24, and 48 h at each temperature (9 different time-temperature conditions). The second method involved exposing the plastics to alkaline conditions with thermal pretreatment. Temperatures that resulted in the greatest 40-day BMP values using the first method were selected for subsequent alkaline-thermal testing at pH values of 8, 10, and 12 and incubation durations of 3, 24, and 48 h (3 pH values at 3 different holding times and 2 different temperatures yielded 18 different pretreatments for each bioplastic).

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For pretreatment, a bioplastic suspension (25 g/L) in deionized water was placed into a 50 mL glass vial or 500 mL glass Erlenmeyer flask. The suspension was mixed with a magnetic stir bar and the pH was increased by sodium hydroxide addition. Thermal pretreatment was done in a water bath continuously mixed at 150 rpm on an orbital shaker (Stuart–Bibby Scientific SBS40 Shaking Water Bath). After thermal pretreatment, the slurry was allowed to cool to ambient temperature and the pH was adjusted to approximately 7 using hydrochloric acid. Pretreated, neutralized bioplastic suspensions were then dried with a laboratory air-blowdown evaporator to facilitate more accurate substrate distribution on a mass basis for anaerobic digestion evaluation.

Untreated and pretreated PHB2 samples were observed by scanning electron microscope (SEM) imaging to visualize the physical effect of thermal alkaline

pretreatment. Surface morphology was captured via JEOL JSM-6510LV SEM imaging (JEOL Ltd., Akishima, Tokyo, Japan) under high vacuum at an accelerating voltage of 20 kV and magnifications of x500 and x5,000 PHB particles were mounted to SEM

specimen mounts with carbon tape and sputter-coated with gold and palladium to a thickness of approximately 200 Å (20 nm).

3.3.3 Biochemical Methane Potential (BMP) Assays

BMP assays were employed to evaluate biomethane yields from untreated and pretreated bioplastics and reported at 40-day test duration unless otherwise noted at 15 or 60 days. BMP assays were performed in triplicate, as described elsewhere (Owen et al., 1979). Briefly, serum bottles (160 mL) were seeded with 50 mL of biomass and 5 mL of bioplastic slurry (25 g/L) containing either pretreated bioplastic, untreated bioplastic as

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negative control (NC), 5 mL of de-ionized water as blank control (BC), or 5 mL of glucose solution (13 g/L) as positive control (PC). Serum bottles were capped with butyl rubber stoppers (Geo-Microbial Technologies, Ochelata, OK) and crimped with

aluminum seals. Setup was performed within a vinyl anaerobic glove box (Coy

Laboratory Products, Grass Lake, MI) purged with nitrogen (N2) gas and less than one

percent hydrogen (H2) gas. BMP assays were incubated (35 °C) with constant orbital

mixing at 150 rpm (New Brunswick Scientific—Model C25KC, Edison, NJ). Serum bottle biogas volume was measured intermittently with wetted glass barrel syringes at ambient pressure and 35 °C, whereas serum bottle headspace methane concentration was determined by gas chromatography. All BMP values were calculated by subtracting the blank control biomethane production value from the BMP gross test value. Lag time was defined as the period between initiation of the BMP assay and the time when the

biomethane production rate exceeded that of the blank control. Seed biomass was a mesophilic (35 °C) laboratory-maintained methanogenic, anaerobic biomass (15.5 ± 0.2 g/L total solids [TS], 7.1 ± 0.2 g/L volatile solids [VS]) fed dry milk substrate (3.5 g/LR-day) and basal nutrient media (Appendix 3, Table 3A) every day with a 15 day solids retention time (SRT) and continuous mixing. Biomass was stored for an average of approximately 1 week at 35 °C in 1 L amber glass jars with loose-fitted lids to allow for gas evolution prior to BMP analyses.

3.3.4 Anaerobic Co-digesters

Synthetic municipal wastewater sludge (SMWS) was digested alone or was co-digested with either untreated or pretreated PHB1 and PHB2 (see Table 3.1 for bioplastic

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abbreviations) in duplicate anaerobic co-digesters (eight digesters total). Co-digesters were 2.5 L bench-scale, continuously stirred-tank reactors (CSTR) operated with a 15-day SRT and 15-15-day hydraulic residence time for 175 15-days. Conditions were maintained at 35.7 °C ± 2.1% and a constant mixing rate of 350 rpm using a magnetic stir bar. Co-digesters were seeded with mesophilic municipal anaerobic biomass (VS = 3.5%) from the South Shore Water Reclamation Facility (Oak Creek, WI). SMWS was composed of basal nutrient media, alkalinity (Appendix 3, Table 3A) and particulate substrate

provided by ground dog food (1.21 ± 0.12 g COD/g dog food) sieved to less than 0.8 mm particle size having approximately 21% protein and 13% fat (Nutro Natural Choice, Franklin, TN, USA). Dry dog food provides a consistent, well-balanced substrate for consistent experimental digesters. SMWS was fed at an organic loading rate (OLR) of 3.6 g COD/LR-day, which was equivalent to 7.5 g dog food/day (Carey et al., 2016). The bioplastic OLR was 0.75 g theoretical oxygen demand (ThOD) per liter of reactor per day (ThOD/LR-d) which was approximately 20% of the COD OLR from SMWS alone. Control digesters were fed SMWS and untreated PHB bioplastic as a co-substrate.

SMWS was fed to all co-digesters without bioplastic from days 1 to 115; subsequently bioplastic was co-fed with SMWS from days 116 to 175. Digester

performance was assessed by daily monitoring of temperature, pH, and biogas production as well as weekly biogas methane content, volatile fatty acids (VFA) concentrations, and solids analysis. Daily biogas volume produced was collected in gas sampling bags (Cole Parmer Kynar PVDF 20.3 L) and subsequently measured with a wet test meter (Precision Scientific). Bench scale anaerobic digestion lag time was defined as the period between day 115 when PHB co-digestion was initiated and the time when the rate of co-digester

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biomethane production exceeded that of the digester fed SMWS alone. Quasi steady-state operation was defined as occurring after all digesters were operated under consistent conditions for at least three SRTs (i.e., 45 days) and biogas production rate values did not vary more than 10%.

3.3.5 Analyses

Biogas was analyzed for methane content by gas chromatography with thermal conductivity detection (GC-TCD) (GC System 7890A, Agilent Technologies, Irving, TX, USA) and data were reported at 35 °C and 1 atm. Total solids (TS), volatile solids (VS), and COD concentrations were measured by standard methods (APHA et al., 1999). VFA concentrations were determined by gas chromatography flame ionization detection (GC-FID) after samples were centrifuged, supernatant filtered through 0.45 µm syringe-tip filter, and acidified with phosphoric acid (Schauer-Gimenez et al., 2010). Since accurate bioplastic COD analysis was not achievable, the bioplastics ThOD values were calculated based on the bioplastic mass and molecular structure, with ratios of 1.67 g ThOD/g PHB and 1.33 g ThOD/g PLA. Bioplastics theoretical maximum methane production values (35 °C, 1 atm) were calculated using the Buswell Equation (Buswell and Mueller, 1952) and were 0.66 L CH4/g PHB and 0.53 L CH4/g PLA. Statistical analyses were performed in R Studio version 3.4.1. Normal distributions were not assumed, and significant

differences among mean BMP values were determined using the non-parametric Mann-Whitney-Wilcoxon test with a confidence level of 0.95 and one-sided alternative hypothesis.

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3.4 Results and Discussion

3.4.1 Bioplastic Pretreatment and BMP Assays

Pretreatment of PHB1 qualitatively resulted in visible surface erosion, increased porosity, and increased surface area compared to untreated (Figure 3.1). Increasing PHB surface area and porosity increases the available binding sites for biological enzymatic degradation and may therefore increase hydrolysis rates (Shang et al., 2012). Hydrolysis of recalcitrant substrates can be the rate-limiting step in methanogenesis, thus

pretreatments that can facilitate increased rates of hydrolysis may increase the rate of methanogenesis (Venkiteshwaran et al., 2016). Thermal alkaline pretreatment of PHB and PLA bioplastics increased anaerobic biodegradability in terms of increased BMP values and reduced lag time compared to untreated controls as described below.

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Figure 3.1 Scanning electron micrographs of untreated and pretreated PHB2 (MirelTM

F1006) after processing. Untreated PHB2 at magnification x500 (top, left) and x5,000 (top, right). Pretreated PHB2 at 500x (bottom, left) and 5,000x (bottom, right),

pretreatment conditions were 90 °C and pH 12 for 48 h.

BMP values and lag times resulting from 27 different pretreatment conditions (i.e., three temperatures at three pH values and three different contact times) for each bioplastic were determined and provided an initial assessment of biomethane production changes due to pretreatments for each bioplastic (see Appendix 3, Table 3B–3F). Percent conversion values for PHB and PLA to biomethane were calculated as the quotient of BMP value divided by the theoretical maximum methane production value determined from the bioplastic ThOD loading. Compared to untreated bioplastics, pretreated PHB

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and PLA resulted in increased average BMP values. The pretreatment conditions resulting in the maximum increases in methane production are presented in Figure 3.2. Maximum percent conversion to biomethane for PHB was 101 ± 6% and 22 ± 6% for PLA after 40 days. Lag times of pretreated PHBs and PLA compared to untreated control digesters were reduced up to 60 and 98%, respectively.

Figure 3.2 BMP values for untreated (gray) and pretreated (black) bioplastics under

conditions resulting in the greatest biomethane increase. The specific conditions are written under each bar in the graph (temperature, pH, duration). BMP values, shown within each bar, with 40 days’ duration are reported at 35 °C and ambient pressure. Percentages above black bars indicate relative increase from untreated to pretreated, with statistically significant differences at 95% confidence denoted by an asterisk (*). Error bars are relative standard deviation (n = 3); some error bars are small and not visible.

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BMP values for pretreated PHBs averaged 360 ± 18 mL CH4/g ThOD (35 °C, 1 atm) representing 91 ± 4% conversion to biomethane, whereas untreated PHBs averaged 270 ± 71 mL CH4/g ThOD and converted 67 ± 19% to biomethane (Figure 3.2). An additional 20 days of BMP analysis yielded averages of 101 ± 4% and 76 ± 17% conversion for pretreated and untreated PHBs, respectively. Pretreatment led to

statistically significant increased BMP values for PHB2 and PHB4, but not for PHB1 and PHB3 (see Appendix 3, Tables 3B–3E). Although the average BMP value of pretreated PHB1 increased by 100% compared to that of the untreated PHB1, the difference was not statistically significant due to high variance in the untreated BMP measurements (RSD ± 81%).

Methane-derived PHB3 exhibited rapid conversion to biomethane at 60 ± 1% after 15 days despite a negligible response to pretreatment. Other reports described untreated PHB conversion to biomethane at 39% in 5 days, 87% in 21 days, 92.5% in 22 days, and 100% in 98 days (Budwill et al., 1996, 1992; Yagi et al., 2014). Individual BMP results from each pretreated PHB vary, but the largest increase in BMP relative to untreated PHB were generally demonstrated at pretreatment conditions of 55 °C, pH value of 12, and 24 or 48 h pretreatment duration, which agrees with reports concluding that abiotic pretreatment of PHB at elevated temperature and pH produced degradation products (Yu et al., 2005).

Compared to untreated PLA, pretreatment of PLA resulted in the largest increase in BMP of the bioplastics studied (Appendix 3, Table 3F). Untreated PLA did not anaerobically degrade to biomethane, whereas pretreatment at 90 °C, pH value at or above 7 for 48 h significantly increased BMP to an average of 79 ± 8 mL CH4/g ThOD

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and equivalent to as much as 22 ± 6% conversion to biomethane. Extending the BMP analysis another 20 days resulted in an additional 5% conversion to biomethane for PLA. Low PLA conversion to biomethane under mesophilic conditions has been reported by others. Kolstad et al. (2012) observed no biomethane evolution in mesophilic anaerobic digesters after 170 days, whereas others reported low conversion to biomethane from 12% at 77 days, 23% at 182 days, and up to 49% after 277 days (Yagi et al., 2014, 2009). In contrast, thermophilic anaerobic digestion of PLA was reported to yield higher rates of digestion with nearly 25% conversion to biomethane in 30 days and up to 75% in 75 days (Yagi et al., 2013). One study attempted pretreatment of PLA at 70 °C for 1 h with no pH control, but this resulted in less biomethane than untreated PLA (Endres and Siebert-Raths, 2011). Results from previous studies are in close accordance with the results herein. However, many of the previous investigations acclimated their seed inocula to enrich for bioplastic fermenting bacteria, whereas the work described herein did not. The BMPs reported herein are for unacclimated biomass that may result in longer lag time and lesser biomethane production within 40 days.

Thermal alkaline pretreatment of bioplastics generally resulted in reduced lag time compared to untreated bioplastics. Average lag time for untreated PHBs was greater than that for pretreated PHB. Untreated PLA did not yield biomethane after 60 days, but pretreated PLA demonstrated no detectable lag time (Figure 3.3). Lag times of untreated PHB3 were longer than those for pretreated PHB2 and highlighted that some commercial PHBs may not anaerobically degrade quickly, especially when using unacclimated biomass. The PHB3 was notable in that pretreatment did not result in a decreased lag time, whereas lag times for all other PHBs and PLA were reduced. In the case of PLA,

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lag time was inversely correlated to pretreatment duration, with pretreatment times of 3, 24, and 48 h resulting in sequentially decreasing lag time of >3 weeks, 2 weeks, and no lag time, respectively (Figure 3.3E). Similarly, Yagi et al. (2009) reported a 55-day lag time for untreated PLA and others reported no anaerobic degradation for untreated PLA (Criddle et al., 2014; Kolstad et al., 2012). Yagi et al. (2014) suggested that mesophilic anaerobic microbial consortia may only have the ability to degrade low molecular weight PLA, and based on the BMP tests conducted here, it is possible that substantial methane production only occurred from low molecular weight PLA produced by thermal

hydrolysis during pretreatments at 90 °C and 48 h. Longer pretreatment duration of PLA correlated to decreased lag time to the point when 48 h of pretreatment eliminated lag time altogether. PLA pretreatment at alkaline pH at 90 °C for durations longer than 48 h may result in increased BMP and potentially complete conversion to biomethane during anaerobic digestion.

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Figure 3.3 Average cumulative biomethane produced during BMP assays (n = 3, error

bars and one standard deviation, 35 °C, ambient pressure) vs. time elapsed for PHB1 (A), PHB2 (B), PHB3 (C), PHB4 (D), PLA (E) after pretreatment. Conditions of pretreatment are denoted on each chart as temperature, °C _ pH _ incubation time, h. Dashed lines show incubation times and pH 8 (), pH 10 (), pH 12 (), and highest biomethane production (♣). Solid lines show controls; negative control (NC •) was untreated bioplastic, positive control (PC ◦) was glucose, straight dotted line denotes theoretical maximum (T) biomethane production, and lag time shown to the right of each chart.

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3.4.2 Bench Scale Co-digestion

Co-digestion of SMWS and PHB was feasible at bench scale as evidenced by efficient biotransformation to biomethane, while pH, temperature, VFAs, and VS

removal remained stable (Table 3.2; Appendix 3, Figures 3A–3C). When bioplastics were co-digested, biomethane production increased 17% over that from digesting SMWS alone. Quasi steady state co-digestion of SMWS and PHB, after 45 days exhibited approximately 80–98% conversion of PHB to biomethane (Table 3.2). Calculations for conversion percentage of bioplastic to biomethane relied upon theoretical biomethane yield.

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34

SMWS Digestion SMWS + PHB Co-Digestion

PHB1_U PHB1_P PHB2_U PHB2_P PHB1_U PHB1_P PHB2_U PHB2_P

Biogasa (L/d) 5.7 ± 0.5 5.6 ± 0.5 5.6 ± 0.5 5.7 ± 0.5 7.0 ± 0.5 6.8 ± 0.7 6.7 ± 0.5 6.6 ± 0.3 pH 7.31 ± 0.02 7.29 ± 0.03 7.29 ± 0.02 7.29 ± 0.02 7.27 ± 0.05 7.24 ± 0.05 7.24 ± 0.04 7.25 ± 0.04 VFA (mg/L) 47 ± 3 51 ± 6 48 ± 5 46 ± 2 47 ± 4 47 ± 4 45 ± 2 45 ± 3 % VSR b 77 ± 1 76 ± 2 77 ± 1 76 ± 1 81 ± 1 78 ± 1 78 ± 1 78 ± 1 % VS 0.69 ± 0.02 0.72 ± 0.02 0.71 ± 0.02 0.73 ± 0.01 0.72 ± 0.02 0.83 ± 0.02 0.81 ± 0.01 0.81 ± 0.01 % CH4 67 ± 3 67 ± 4 68 ± 4 67 ± 4 65 ± 0.4 64 ± 0.7 65 ± 0.4 66 ± 0.6

a Average and standard deviation values from duplicate digesters b Percent volatile solids reduction (VSR) from feedstock to effluent

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Average pH of digester effluent fed SMWS alone was 7.30 ± 0.02, while pH in all digesters dropped slightly after PHB was fed to the digesters. The pH difference was statistically significant during quasi steady state co-digestion with PHB at an average value 7.24 ± 0.02 (Appendix 3, Figure 3A). VFA concentrations of digester effluent expressed as acetic acid equivalents were 48 ± 4 mg/L and 46 ± 3 mg/L before and during co-digestion at quasi steady state for all digesters, respectively, and were not statistically different (Appendix 3, Figure 3B). The VS as a percent of TS in digester effluent deviated only 2% for all digesters and ranged between 57 and 59% (Appendix 3, Figure 3C).

The VS reduction (VSR) values increased for all digesters when PHB was co-digested and the average increased from as low as 75 ± 1% during SMWS digestion alone to as much as 81 ± 1% when bioplastic was co-digested. Solids initially increased in response to PHB addition but attained a quasi- steady state value after 15 days or one SRT. Average percent biomethane in biogas decreased from 2 to 3% when PHB was co-digested (Table 3.2), but the differences were not statistically significant.

In contrast to co-digestion of untreated PHB, co-digestion of pretreated PHB increased biomethane production by 5% and reduced lag time by approximately 4 days for both PHB1 and PHB2 (Figure 3.4). Lag time for bench scale co-digestion of PHB2 was 6 days for untreated and 3 days for pretreated bioplastic.

PHB co-digestion with synthetic primary sludge increased both the overall rate and extent of biomethane production compared to anaerobic digestion of synthetic primary sludge alone (Figure 3.4).

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36 Figure 3.4 Daily biomethane production for continuously fed anaerobic digesters (n = 2, error bars show standard deviation)

comparing (top, left) untreated PHB1, (bottom, left) pretreated PHB1 (treatment: 55 °C, pH = 12, 24 h) and (top, right) untreated PHB2, (bottom, right) pretreated PHB2 (55◦C, pH = 12, 48 h). Quasi steady-state was assumed after 45 days with average biomethane production (L/d) at quasi steady state presented in parentheses. Solid lines depict gas production rates before and after PHB

co-digestion, dotted lines show theoretical co-digestion production based on 40 days BMPs. Solid arrows proportionately illustrate average lag period (d) between PHB addition and increased biomethane production. Steady state conversion of PHB to biomethane (%) and higher heating value of methane per kg PHB was based on an expected 21% increase in biomethane yield.

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3.5 Conclusions

Biodegradable bioplastic can be co-digested under stable conditions at municipal water resource recovery facilities to generate renewable energy. Bioplastic pretreatment (≥55◦C, pH ≥ 10, ≥24 h) resulted in more rapid and complete anaerobic bioplastic co-digestion. With pretreatment, partial anaerobic digestion of PLA was accomplished. In addition, thermal alkaline bioplastic pretreatment reduced lag time before biomethane production occurred and increased bioplastic conversion to biomethane. Pretreatment of PHB bioplastic under quasi steady state co-digestion conditions resulted in approximately 5% greater biomethane production compared to untreated PHB. Bioplastic co-digestion at the loadings used increased biomethane production by 17%.

3.6 Acknowledgments

The authors thank the Water Equipment and Policy Research Center (NSF Grant number 1540010) for funding, Mango Materials for providing PHB1, PHB2, and PHB3 and the Virginia Institute of Marine Science for providing PHB4.

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